Endothelial nitric oxide synthase (eNOS) is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms. We have found that eNOS is associated with the actin protein and that this association increases eNOS activity. We propose a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Aim #1: Determine whether actin interacts with the putative actin binding sequences (ABSs) in the eNOS protein resulting in increased electron transfer and catalytic activity of eNOS. We will use a yeast two-hybrid system and the peptides with sequences of the three putative ABSs of eNOS to pinpoint the actin-binding site in the eNOS protein. We will evaluate the effects of ABS peptides of eNOS on eNOS activity, eNOS-actin interaction, the enzyme kinetics, and on cytochrome c and ferricyanide reductase activities in the mixture of purified eNOS and actin to clarify the mechanism for increased eNOS activity by actin association. Aim #2: Determine whether alterations of eNOS activity in lung endothelium exposed to different oxygen tensions are due to changes in eNOS-actin association. We will manipulate the availability of actin in endothelium using siRNA and adenovirus gene transfer technology and introduce the ABS peptides of eNOS into endothelial cells to block eNOS-actin interaction using microinjection technique. Then we will observe hypoxia/hyperoxia-induced alterations in eNOS activity, the actin cytoskeleton, eNOS-actin association, NO release, and endothelium-dependent vasorelaxation. The successful completion of these aims will advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This proposal is to study a novel hypothesis that eNOS-actin association regulates electron transfer of eNOS resulting in increased catalytic activity and that alterations in eNOS-actin interaction mediate the regulation of eNOS in lung endothelium exposed to different oxygen tensions. Proof of the role of eNOS-actin interaction in hypoxia/hyperoxia-induced alteration of eNOS function would advance our understanding of the biology of NO, and in turn, have major implications for pulmonary diseases characterized by impaired eNOS activity and vascular NO production, such as primary and secondary pulmonary hypertension, COPD, and cor pulmonale. Determination of the effect of short-term hyperoxia will help to advance our understanding of the mechanism of oxygen therapy and will lead to better management strategy for patients receiving supplemental oxygen therapy. [unreadable] [unreadable] [unreadable]